Refolding Record:

Protein See all refolding records for this protein...
Protein Name	Beauveria bassiana Chitinase
Abbreviated Name	Bbchit1
SCOP Family	Unknown
Structure Notes
Organism	Beauveria bassiana
UniProt Accession	Q8J1Y3
SCOP Unique ID	n/a
Structure Solved
Class	Unknown
Molecularity	Unknown

Construct
Full Length	y
Domain	n/a
Chimera	n/a
Variants	n/a
Chain Length	349
Molecular Weight	36736.9
Pi	6.19
Molecular Weight	36736.9
Disulphides	Unknown
Full Sequence	MAPFLQTSLALLPLLASTMVSASPLAPRAGTCATKGRPAGKVLQGYWENWDGAKNGVHPPFGWTPIQNPDIRKHGYNVINAAFPIIQPDGTALWEDGMDTGVKVASPADMCEAKAAGATILMSIGGATAAIDLSSSAVADKFVSTIVPILKKYNFDGIDIDIESGLTGSGNINTLSTSQTNLIRIIDGVLAQMPANFGLTMAPETAYVTGGTITYGSIWGSYLPIIKKYLDNGRLWWLNMQYYNGEMYGCSGDSHKAGTVEGFIAQTDCLNKGLSIQGVTITIPYDKQVPGLPAQPGAGG GHMSPSNVAQVLSHYKGALKGLMTWSLNWDGSKNWTFGDNVKGTLGTA
Notes	n/a

Expression
Report	Fan Y, Zhang Y, Yang X, Pei X, Guo S, Pei Y. (2007) Protein Expression and Purification, 56, 93-99
Project Aim	Recombinant Protein Expression
Fusion	None
Protein Expression and Production	Protein recombinantly expressed as and refolded from inclusion bodies.
Expression Host	Escherichia coli
Expression Strain	BL21(DE3)
Expression Temp	37.0
Expression Time	2 h
Expression Vector	pET28a
Expression Protocol	Expression and purification of Bbchit1 in E. coli The intact coding region of Bbchit1 was obtained by PCR with primers P1 and P2 (P1: 5′-gccatggctccttttcttcaaacc-3′, P2: 5′-gaattcttacgcagtccccaaagtccc-3′; the underline sequences were NcoI and EcoRI sites, respectively) using B. bassiana genomic DNA as template. There is no intron in this gene [9]. The PCR product was cloned into pMD-T vector for DNA sequencing. Bbchit1 gene excised from pMD-T vector by NcoI and EcoRI was inserted into the corresponding sites of pET28a. BL21 (DE3) cells were transformed with the recombinant plasmid and pET28a plasmid, respectively. The latter was used as a negative control. The expression of Bbchit1 was followed the guidelines of Novagen. A 50 μl overnight culture of BL21 carrying the recombinant plasmid was inoculated into 15 ml LB medium supplemented with 50 μg/ml kanamycine. The culture was grown at 37 °C until the OD600 reached 0.4 and induced by adding isopropylthio-d-galactoside (IPTG)1 at a concentration of 0.1 mM for 2 h. To determine the effect of IPTG concentration on expression level of Bbchit1, BL21 cells contained pET28a-Bbchit1 plasmid was induced 2 h at a concentration of IPTG, 0.05, 0.1, 0.5, 1, and 2 mM, respectively. To determine the time course of Bbchit1 expression, aliquots of the culture were removed just before IPTG (0.1 mM) induction and 0.5, 2, 4, 6, and 8 h after induction. After induction, the cells were lysed in SDS–PAGE sample buffer at 100 °C for 10 min and the lysates were analyzed by SDS–PAGE. The expression amount and purity of protein was determined from the images obtained with the ImageMaster VDS-CL system (Amersham Biosciences) by Total Lab 1D software.
Method of Induction	IPTG
Cell Density at Induction	OD n/a = n/a
Cell Disruption Method	Freeze-thaw
Lytic Agent	Lysozyme
Pre-Refolding Purification	None
Solubility	insoluble

Refolding
Refolding Method	Dialysis
Wash Buffer	0.5% Triton X-100, 10 mM EDTA, pH 8.0, and 1 mM PMSF
Solubilization Buffer	8 M urea
Refolding Buffer	20 mM Tris–HCl (pH 8.5) and 0.05% Triton X-100
Pre-Refolding Purification	None
Tag Cleaved	no tag
Refolding pH	8.5
Refolding Temperature	25.0
Protein Concentration	n/a
Refolding Time	n/a
Redox Agent	None
Redox Agent Concentration	n/a
Refolding Protocol	Bbchit1 was refolded through dialyzed once against 20 mM Tris–HCl, pH 8.5, and 0.05% Triton X-100 and three against 20 mM Tris–HCl, pH 8.5. Proteins in the polyacrylamide were stained with Coomassie brilliant blue G-250.
Refolding Assay	Activity assay
Refolding Chaperones	None
Refolding Additives	Triton X-100
Additives Concentration	0.05%
Refolding Yield	50 mg per liter
Purity	n/a
Notes	n/a

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